Search

It has recently been documented that, compared to untransformed controls, the roots of oilseed rape (Brassica napus L. CV CrGC5) seedlings transformed by Agrobacterium rhizogenes A4 show a reduced gravitropic reaction (Legue et al. 1994, Physiol Plant 91: 559-566). After stimulation at 90 degrees C or 135 degrees, the transformed root tips curve. but never reach a vertical orientation. In the present study, we investigated the causes of reduced gravitropic bending observed in stimulated transformed root tips. First, we localized the gravitropic curvature in normal and in transformed roots after 1.5 h of stimulation. The cells involved in root curvature (target cells) corresponded at the cellular level to the apical part of the zone of increasing cell length. In transformed roots grown in the vertical position, these cells showed a reduction in cell length compared to controls. Because auxin is considered to be the gravitropic mediator, the response of normal and transformed roots to exogenous auxin was studied. Indole-3-acetic acid (IAA) was applied along the first 3 mm using resin beads loaded with the hormone. In comparison to normal roots, transformed roots showed reduced bending toward the bead at all points of bead application. Moreover, the cells which responded to IAA corresponded to the target cells involved in the gravitropic reaction. The level of endogenous IAA was lower in transformed roots. Thus, it was concluded that the modified behavior of transformed roots during gravitropic stimulation could be due to differences either in IAA levels or in reactivity of the target cells to the message from the cap.

Transgenic hairy roots of Brassica napus (cv. Omega) have been developed, using Agrobacterium rhizogenes strain AR 25, for use as a model system in the investigation of physiological and morphological differences between transgenic and normal roots. The basic parameters of growth and normal or altered gravitropical behaviour of hairy roots are for the first time presented in this paper together with an ultrastructural and morphological analysis of the root statocytes. The results obtained also represented the basis for the TRANSF0RM-experiment on the IML-2 mission performed onboard the Space Shuttle Columbia. Typical hairy root traits such as hormone-autonomous growth high growth rate, lateral branching, and changed/absence of gravitropism were detected. The transformed nature of the roots was confirmed by Southern blot analyses. The gravitropical behaviour of apices from hairy root cultures of this clone has been compared with root tips from normal seedlings. While the wild type roots curved progressively with increasing stimulation angles, the transformed roots showed no curvature when stimulated at 45 degrees, 90 degrees or 135 degrees on the ground. The morphology and ultrastructure of the root tip regions were examined by light microscopy and transmission electron microscopy. At the ultrastructural level no major differences could be detected between the roots studied. There was, however, a slight reduction in the starch content of most of the amyloplasts of the transgenic root tips, and the root cap was more V-shaped in the transgenic roots than in the wild type. Preliminary results from the Shuttle experiment TRANSFORM show a random distribution of amyloplasts in the root cells of both transformed and wild type root caps after 14 h on a 1xg centrifuge followed by 37 h in microgravity.

The initial event of gravity perception by plants is generally thought to occur through sedimentation of amyloplasts in specialized sensory cells. In the root, these cells are the columella which are located toward the center of the root cap. To define more precisely the contribution of columella cells to root gravitropism, we used laser ablation to remove single columella cells or groups of these cells and observed the effect of their removal on gravity sensing and response. Complete removal of the cap or all the columella cells (leaving peripheral cap cells intact) abolishes the gravity response of the root. Removal of stories of columella revealed differences between regions of the columella with respect to gravity sensing (presentation time) versus graviresponse (final tropic growth response of the root). This fine mapping revealed that ablating the central columella located in story 2 had the greatest effect on presentation time whereas ablating columella cells in story 3 had a smaller or no effect. However, when removed by ablation the columella cells in story 3 did inhibit gravitropic bending, suggesting an effect on translocation of the gravitropic signal from the cap rather than initial gravity perception. Mapping the in vivo statolith sedimentation rates in these cells revealed that the amyloplasts of the central columella cells sedimented more rapidly than those on the flanks do. These results show that cells with the most freely mobile amyloplasts generate the largest gravisensing signal consistent with the starch statolith hypothesis of gravity sensing in roots.

Microsomal and soluble protein fractions from Vicia faba root tips were used for SDS-PAGE and Western-immunoblot analysis with anti-ubiquitin antibodies after 9 h clinostat treatment of the plants. In contrast to soluble proteins omnilateral gravistimulation (9 h) resulted in an enhanced proteolytic capacity for microsomal proteins. The increase of vanadate-sensitive ATPase activity was 83% after 9 h clinostat treatment, when the enzyme activity was measured directly after membrane preparation. Enhanced ATPase activity was correlated with the appearance of a polypeptide of about 100 kDa and its fragments (93 and 80 kDa). ATPases are not the only membrane bound proteins, which are changed during clinostat treatment, as several ubiquitinated polypeptides were also affected. A 1 h storage of microsomal fractions led to a shift of band intensities on ubiquitin-specific Western-blots. The demonstrated effect could not be observed, when fractions were isolated in the presence of protease inhibitors. In accordance with the polypeptide analysis omnilateral gravistimulation resulted in an enhanced capacity to degrade specific microsomal ubiquitin-conjugates, whereas the soluble ubiquitin-pool was not visibly affected.

When statocytes of cress root-caps were incubated in an extraction medium containing phalloidin, bundles of microfilaments (6.6 +/- 2.4 nm in diameter) were found in the cortical cytoplasm. The inclusion of heavy-meromyosin in this medium demonstrated an arrowhead pattern on the filaments indicative of the presence of actin.